We have cloned part of the human 25-OHD 24-hydroxylase cytochrome P450 (P450cc24) cDNA. The characterized sequence consists of 776 bp of the coding and 720 bp of the 3'-untranslated region interrupted by an intron. In the coding region we found 79.8% similarity in DNA and 87.5% in deduced amino acid sequences between human and rat, with no similarity in the 3'-untranslated region. By Southern blot hybridization of DNA from human-hamster somatic cell hybrids and by in situ immunofluorescence hybridization, we mapped P450cc24 to human chromosome 20q13.1. This location of P450cc24 is different from that of pseudovitamin D-deficient rickets (PDDR), previously assigned to chromosome 12q14 by linkage analysis, thus excluding it as a target of the PDDR mutation. Since it is likely that PDDR is caused by a mutation in the 25-OHD 1 alpha-hydroxylase P450 subunit (P450cc1 alpha) our results do not support the hypothesis that the two cytochromes are encoded by a single gene.